We previously showed that testosterone, administered in vivo, increases the tone of cerebral arteries. A possible underlying mechanism is increased vasoconstriction through the thromboxane A2 (TxA₂) pathway. Therefore, we investigated the effect of chronic testosterone treatment (4 wks) on TxA₂ synthase levels and the contribution of TxA₂ to vascular tone in rat middle cerebral arteries (MCA). Using immunofluorescence and confocal microscopy, we demonstrated that TxA₂ synthase is present in MCA segments in both smooth muscle and endothelial layers. Using Western analysis, we found that TxA₂ synthase protein levels are higher in cerebral vessel homogenates from testosterone-treated orchiectomized (ORX+T) rats as compared to orchiectomized (ORX) control animals. Functional consequences of changes in cerebrovascular TxA₂ synthase were determined using cannulated, pressurized MCA segments in vitro. Constrictor responses to the TxA₂ mimetic, U-46619, were not different between the ORX+T and ORX groups. However, dilator responses to either the selective TxA₂ synthase inhibitor, furegrelate, or the TxA₂-endoperoxide receptor (TP) antagonist, SQ-29548, were greater in ORX+T compared to ORX. In endothelial-denuded arteries, the dilation to furegrelate was attenuated in both ORX and ORX+T, and the difference between the groups was abolished. These data suggest that chronic testosterone treatment enhances TxA₂-mediated tone in rat cerebral arteries by increasing endothelial TxA₂ synthesis without altering the TP receptors mediating constriction. The effect of in vivo testosterone on cerebrovascular TxA₂ synthase, observed here following chronic hormone administration, may contribute to the risk of vasospasm and thrombosis related to cerebrovascular disease.